Shota Yamashita (Postdoctoral Fellow, National Institute of Genetics / 3rd year of Doctoral program, Department of Biological Sciences (at the time of research))

Hisayoshi Nozaki (Project Researcher / Associate Professor, Department of Biological Sciences)

Key points of the presentation

• We have sequenced and analyzed the whole genome of the multicellular green alga Astrephomene ^{(Note 2), which} has acquired somatic cells independently of Volvox ^{(Note 1)}, and analyzed genes involved in somatic cell differentiation.
• We found that the somatic gene expression patterns of both species are similar, despite the fact that Astrephomene does not have the somatic differentiation genes that have been identified in Borrelia volvox.
• It is hoped that a more detailed comparison of somatic cell evolution in Astrephomene and Volvox will elucidate the universal principles of the evolution of somatic cell acquisition in multicellular organisms.

Summary of Presentation

The bodies of multicellular organisms such as animals and plants are composed of many cells, but only a few cells produce the next generation, and most are inevitably destined to die as somatic cells, which is considered to be "senescent death," the fate of multicellular individuals. However, how such somatic cells were acquired during the evolution from unicellular to multicellular organisms is largely unknown. Only in the green alga Volvox was a gene involved in somatic cell differentiation discovered and its evolution studied.

The University of Tokyo Graduate School of Science, the National Institute of Genetics, the National Institute for Environmental Studies, and other research groups conducted whole-genome sequencing and other analyses of Astrephomene, which has evolved very few somatic cells, independently of Volvox, and found that Astrephomene does not have the somatic differentiation genes of Volvox, and that Astrephomene has a pattern of somatic cell expression despite this lack of somatic cell differentiation genes. The results showed that Astrephomene lacks the somatic differentiation genes of Volvox, and that the gene expression patterns of differentiated somatic cells are similar between the two species. This study also identified a candidate somatic differentiation gene that evolved independently in Astrephomene. It is expected that a more detailed comparison of somatic cell evolution in Astrephomene and Volvox will elucidate the universal principles of the evolution of somatic cell acquisition in multicellular organisms, which could not be elucidated only by the findings in Volvox so far.

Contents of Presentation

We humans, animals, and plants are multicellular organisms whose bodies are composed of many cells. Among the cells that make up the bodies of multicellular organisms, only a few cells produce the next generation as reproductive cells, and most of the remaining cells inevitably die as somatic cells, which leads to aging and death of multicellular individuals. ^{(Note 3)} On the other hand, in unicellular organisms, all cells have the potential to reproduce by division and become the next generation, and no cells inevitably die through generation. Thus, the role differentiation between germ cells and somatic cells that leads to "senescent death" in multicellular individuals has been independently acquired many times during the process of evolution from ancestors that were unicellular organisms to multicellular organisms. However, it has remained largely unclear what genes have evolved during the evolutionary process to acquire somatic cell differentiation.

The organisms that hold the key to solving this evolutionary mystery are the Volvox series of green algae (Fig. 1). The Volvox lineage includes species with a wide range of intermediate evolutionary traits, from the unicellular organism Chlamydomonas to the simple multicellular organism Volvox, a collection of about 2000 cells (Fig. 1A). The somatic and germ cell differentiation is particularly well suited to the study of the evolution of multicellular organisms acquiring somatic cell differentiation, as it includes both species in which all cells reproduce without somatic cells (e.g., Gonium, Fig. 1B) and those in which all cells have somatic cell differentiation (e.g., volvox, Fig. 1C).

Somatic cells in volvox are small and outwardly aligned, specializing in flagellar motility. The inner germ cells, on the other hand, do not have flagella and are specialized for growth and subsequent reproduction through photosynthesis. The somatic cells provide the energy for swimming as multicellular organisms, allowing the germ cells to focus their energy on their own reproduction (Fig. 1C). A transcription factor called regA^{(Note 4)} is responsible for this differentiation, and regA is expressed only in somatic cells to control their differentiation fates ( Ref. 1). However, the only example of somatic cell evolution that has been elucidated at the genetic level is the Borbox regA, and our knowledge is insufficient to elucidate the universal principles of somatic cell evolution, which is widely observed in multicellular organisms.

Figure 1: A. Phylogenetic relationships and somatic cell evolution of the Volvox lineage (based on genome phylogenetic analysis in this study). The Volvox-series green algae are a group of organisms that includes a wide range of species with intermediate traits, from the unicellular Chlamydomonas to the Volvox with somatic and germ cell differentiation. In recent years, whole genomes of representative species have been sequenced one after another, and evolutionary biology of multicellularity has been studied based on comparative genome analysis. In this group, somatic cell acquisition has occurred independently in the ancestors of Borrelia volvox and Astrephomene. B. The life cycle of Gonium, which forms an ancestral multicellular body without cell differentiation. In Gonium, all cells reproduce asexually and form daughter clusters. In C. volvox, somatic cells and germ cells are differentiated, and the germ cells reproduce asexually to form a daughter population, while somatic cells do not reproduce and are destined to die.

Therefore, we focused on Astrephomene, another green alga in the Volvox family. Astrephomene has acquired somatic cells independently of Volvox (Fig. 1A), and is characterized by the formation of only four small somatic cells at the back end of a multicellular body consisting of 64 cells (Fig. 2). The extremely small percentage of somatic cells and the fact that both somatic cells and germ cells have flagella, with the only difference being cell size, make astrephomene an interesting organism even as a very early stage of somatic cell differentiation.

Figure 2: Astrephomene, an algae that evolved somatic cells independently from the volvox. a. Surface view of an Astrephomene multicellular body. b. Median section of an Astrephomene multicellular body. c. Cross-section of an Astrephomene multicellular body. C. Four somatic cells of Astragalus astrephomene. D. Astragalus astrephomene in the process of asexual reproduction. The germ cells are undergoing cell division and embryogenesis, but the somatic cells (arrowheads) do not reproduce asexually. The photos A-C are reproduced from Ref. 2.

The University of Tokyo's Graduate School of Science, the National Institute of Genetics, the National Institute for Environmental Studies, and other research groups have recently used a newly established culture strain of Astrephomene ( Reference 2) to decode the entire genome of Astrephomene. The genomic data of Astrephomene did not reveal any regA genes, suggesting that Astrephomene evolved somatic cells by acquiring regulatory factors different from those of Borrelia volvox.

In addition, by developing a method to separate somatic cells from germ cells (Fig. 3A), we performed cell-specific RNA-seq analysis of Astrephomene ^{(Note 5)} and compared gene expression patterns between somatic cells and germ cells. The results revealed that the expression of some genes involved in flagellar motility was upregulated in somatic cells, while the expression of genes involved in photosynthesis and assimilation pathways was downregulated compared to germ cells. This was similar to the gene expression pattern in somatic cells of the volvox ( Ref. 3). In addition, the MYB transcription factor ^{(Note 6)} and the RWP-RK transcription factor ^{(Note 7),} which are expressed only in somatic cells, were found to be candidates for regulators of somatic cell differentiation in astrephomene. In other words, while different genes evolved in Volvox and Astrephomene for the regulation of somatic differentiation, the regulated gene expression patterns were shown to be common (Figure 3B).

Figure 3: Cell-specific RNA-seq analysis of astrephomene. a. Method developed in this study to separate somatic and germline cells of astrephomene. A. Method developed in this study to separate somatic and germ cells of Astrephomene. B. Gene expression patterns of cell differentiation in Astrephomene revealed by cell-specific RNA-seq analysis. and a schematic diagram of the commonalities between the two and the volvox. The size of the letters indicates the magnitude of gene expression. In somatic cells of Astrephomene, the expression of some genes involved in flagellar motility was up-regulated, while the expression of genes involved in photosynthesis and assimilation pathways was down-regulated compared to germline cells. This trend is similar to that in volvox. While regA is responsible for this differentiation in the volvox, the MYB transcription factor and the RWP-RK transcription factor, which are expressed only in somatic cells, were found to be candidate regulators for this function in astrephomene. Based on the results of this study.

The results of this study suggest that the four somatic cells of Astrephomene, like the somatic cells of Volvox, are destined to die without reproducing by suppressing growth through photosynthesis, and instead help other cells reproduce by specializing in motility. This is an interesting result in astrephomene, which also swims with flagella, and more detailed studies of the functions of the four somatic cells in astrephomene may reveal the minimum requirements for a multicellular body to require somatic cells. On the other hand, different genes evolved in both species as regulators of somatic cell differentiation, indicating that diverse genes may have been involved in the evolution of somatic cells during the evolution of multicellular organisms. Further research using the genomic data established in this study is expected to identify regulators of somatic differentiation from the candidate genes discovered in Astrephomene and compare their evolution with that of Volvox, thereby elucidating commonalities and general rules in the evolution of somatic cells.

This work was supported by Grant-in-Aid for Scientific Research "Advanced Genome Support" (16H06279) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by Grant-in-Aid for Scientific Research (17J03439 from Shodai Yamashita; 16H02518, 20H03299 from Hisayoshi Nozaki) from the Japan Society for the Promotion of Science.

References
1. Kirk, M. M. et al. regA, a Volvox gene that plays a central role in germ-soma differentiation, encodes a novel regulatory protein. Development 1999; 126.
2. Yamashita, S. et al. Alternative evolution of a spheroidal colony in volvocine algae: Developmental analysis of embryogenesis in Astrephomene ( Volvocales, Chlorophyta). BMC Evol. Biol. 2016; 16:243.
3. Matt, G. Y. & Umen, J. G. Cell-type transcriptomes of the multicellular green alga Volvox carteri yield insights into the evolutionary origins of germ and somatic differentiation programs. Genes Genomes Genetics 2018; 8:531-550.

Journal of Publication

Journal Name.	Scientific Reports
Article title.	Genome sequencing of the multicellular alga Astrephomene provides insights into convergent evolution of germ-soma differentiation .
Author(s)	ShotaYamashita, KayokoYamamoto, Ryo Matsuzaki, Shigekatsu Suzuki, HaruyoYamaguchi, Shunsuke Hirooka, Yohei Minakuchi, Shin-ya Miyagishima, Masanobu Kawachi, Atsushi Toyoda and Hisayoshi Nozaki
DOI Number	10.1038/s41598-021-01521-x

Terminology

1 Volvox (scientific name: Volvox )

A swimming green alga that forms spherical multicellular bodies consisting of several hundred to several thousand cells. It is a so-called phytoplankton and is commonly found in rice paddies and lakes from spring to summer. It has been used in research as a model for simple multicellular organisms because of the differentiation of somatic cells and germ cells. ↑up

Note 2: Astrephomene (scientific name)

A swimming green alga that forms spherical multicellular bodies consisting of 32 or 64 cells. It is characterized by the formation of only four somatic cells at the posterior end of the multicellular body. It also has many interesting features in the evolution of green algae in the Volvox family, such as a different mechanism for forming spherical cell layers during embryogenesis (Ref. 2), and a dependence on organic matter in the environment for growth, although it also engages in photosynthesis. It is found in rice paddies, but is a rare species and had not been reported from Japan until 1983. ↑up

Note 3 Somatic cells and reproductive cells

In this section, germ cells are defined as cells that reproduce (either sexually or asexually) to produce the next generation of individuals, and somatic cells (or non-germ cells) as cells that do not reproduce. In general, somatic cells have the function of assisting the reproduction of germ cells. In the human body, sperm and eggs are germ cells, and all other cells are somatic cells. While somatic cells are intricately differentiated in animals and plants, simple differentiation between somatic cells and germ cells is widely observed in multicellular organisms of various lineages. The death of somatic cells, which inevitably comes in the life cycle, is the "aging death" of a multicellular individual. ↑up

Note 4 regA

A transcription factor that is expressed only in somatic cells of volvox and regulates somatic cell differentiation. regA mutants grow somatic cells and initiate asexual reproduction like germ cells, suggesting that regA suppresses photosynthetic growth and reproduction of somatic cells. It is speculated that it was created by duplication of an ancestral gene that Chlamydomonas and Gonium also have during the evolution of Volvox. ↑up

Note 5 RNA-seq analysis

A method to obtain the expression levels of all genes in the genome in a cell or tissue by extracting RNA from the cell or tissue, creating a library, and decoding it by a next-generation sequencer. In this study, RNA-seq analysis was performed on isolated somatic cells and germ cells, and by comparing the obtained data, genes differentially expressed between somatic and germ cells were detected. ↑up

6 MYB transcription factors

A group of transcription factors. Originally discovered as oncogenes, they are involved in cell cycle regulation in animals. On the other hand, plants have many MYB transcription factors in their genomes and their functions are diversified. ↑up

Note 7 RWP-RK transcription factor

A group of transcription factors with a DNA-binding domain called RWP-RK domain. Known genes include those involved in nitrogen metabolism and MID/OTOKOGI involved in sex determination. ↑up